RESUMEN
Phenol soluble modulins (PSMs) are small amphipathic peptides involved in a series of biological functions governing staphylococcal pathogenesis, primarily by facilitating the formation of an extracellular fibril structure with amyloid-like properties. This fibrillar architecture stabilizes the staphylococcal biofilm making it resilient to antibiotic treatment. Our study aims to abrogate the amyloid fibrillation of PSM α1 with novel insights on the amyloid modulatory potential of a prenylated chalcone, Isobavachalcone (IBC). A combination of biophysical and computational assays to address the amyloid modulatory effect of IBC has been undertaken to arrive at a model for the inhibition of PSM α1 fibrillation. ThT kinetics studies indicated that IBC must be stably interacting with the amyloidogenic core of PSM α1 monomers or it may be inhibiting the pre-fibrillar aggregates populated at the early stages of amyloid transformation kinetics. This heteromolecular association further inhibits the amyloid transformation corroborated by a â¼ 94% and â¼ 91% reduction in the ThT maxima, even at sub-stoichiometric concentrations. Transmission electron microscopy (TEM) of end-stage aggregates (â¼ 55 h) depict mature, inter-twined, laterally stacked amyloid fibrils in untreated PSM α1 samples while this fibrillar load is remarkably reduced in the presence of IBC. The inhibitory effect of IBC on the ß-sheet transitions of PSM α1 were also validated using far-UV CD spectra. Molecular dynamics simulation studies with PSM aggregates (PSM-A) have also suggested that IBC disrupts the hydrogen bonding interactions and corroborates the inhibition of alpha to beta transitions of PSM-A. Collectively, our data proposes a novel structural motif for the rational discovery of non-toxic therapeutic agents targeting the functional amyloids which have slowly emerged as potent factors, consolidating the antibiotic resistant staphylococcal biofilm assembly.
Asunto(s)
Amiloide , Chalconas , Staphylococcus aureus , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/metabolismo , Chalconas/farmacología , Chalconas/química , Chalconas/metabolismo , Amiloide/metabolismo , Amiloide/química , Simulación de Dinámica Molecular , Cinética , Prenilación , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Biopelículas/efectos de los fármacos , Toxinas BacterianasRESUMEN
A widely used method to obtain tetrazoles is through the azide and nitrile [3+2] cycloaddition. However, this process often involves using non-recyclable transition metals or Lewis acid catalysts and stoichiometric amounts of oxidants and additives, which reduces atom efficiency. We have discovered a convergent paired electrochemical reaction to perform this cycloaddition reaction, without the need for metal catalysts or oxidants. This tetrazolation strategy uses azidotrimethylsilane (TMSN3) and N-heterocycles in an undivided cell at a constant current. We use a mixture of CH3CN and equivalent amounts of H2O as co-solvent at room temperature. It is crucial to produce a stoichiometric amount of active hydroxyl ions through the cathodic reduction of water. Cyclic voltammetry (CV) studies and control experiments confirm that the cycloaddition reaction is specific to the electrode electron transfer process, eliminating the need for a mediator to shuttle electrons. This metal- and oxidant-free strategy is highly compatible with different functional groups and produces products with moderate to good yields. We have successfully tetrazolated bioactive compounds at a late stage, scaled up batches efficiently, and synthesized free amino-containing N-heterocycles via denitrogenation of tetrazoles.
RESUMEN
An electrooxidative C-H functionalization is a widely accepted route to obtain sulfur-containing arenes and heteroarenes. However, this process often involves using non-recyclable supporting electrolytes, (co)solvents like hexafluoroisopropanol, additives like acid, or catalysts. The use of additional reagents can increase costs and waste, reducing atom efficiency. Moreover, unlike other nitrogen-containing heterocycles, there have only been sporadic reports of electrochemical C-H functionalization in fused pyrimidin-4-ones, and an electrolyte-free process has yet to be developed. This work demonstrates that such anodic coupling reactions can be performed in an all-green electrolytic system without using such additional electrolytes or HFIP, maintaining a high atom economy. This C-H functionalization strategy utilizes inexpensive sodium sulfinates and ammonium thiocyanate as sulfonylating and thiocyanating agents in an undivided cell at a constant current, using a mixture of CH3 CN/H2 O as solvent at room temperature. Thus, fused pyrimidin-4-ones can be selectively converted into C3-sulfonylated and -thiocyanated derivatives in moderate to good yields.
RESUMEN
BACKGROUND: A major cause of economic losses in the medical implant sector has been bacterial biofilms due to their ability to persist on various surfaces and their tolerance against endogenous defences, antibiotics, or other anti-microbial agents. The quest for potential sources causing inhibition or disruption of bacterial biofilms has been taken up to alleviate the loss. Plantderived extracts such as essential oils, bioactive compounds and other solvent extracts are regularly being used instead of antibiotics and other synthetic compounds as they are safer, economical, and in many instances, have an elaborate history of traditional medicinal usage. OBJECTIVES: As a plant that has been traditionally used over the centuries, the Moringa oleifera Lam., or more commonly the drumstick tree, is being tapped for myriad pharmaceutical applications. The protein-rich leaf of this tree has not only proved to be of great nutritional value but also divulged numerous potential therapeutic applications. METHODS: While reports of proteinaceous components are rare, here we report the efficacy of the aqueous extract of the leaf of M. oleifera and a 62 kDa protein component in the disruption of staphylococcal biofilms, which are largely implicated in nosocomial infections. RESULTS: The application of the M. oleifera leaf extract protein had a marked effect on the biofilm growth or formation by Staphylococcus aureus. CONCLUSION: While the crude extract itself showed considerable disruption of biofilm formation, the application of the purified protein obtained after a two-step process led to a significant increase in the anti-biofilm activity.
Asunto(s)
Antiinfecciosos , Moringa oleifera , Staphylococcus aureus , Extractos Vegetales/farmacología , Antibacterianos/farmacología , Antiinfecciosos/farmacología , Biopelículas , Hojas de la PlantaRESUMEN
Bacteriophage Phi11 harbors a gene, gp13, encoding the putative SSB protein (GenBank accession no. NC_004615.1). SSB proteins bind to and protect the single-stranded DNA molecules from nuclease digestion and are essential for the growth and metabolic activities of the organisms encoding them. In this investigation, we have carried out the cloning, recombinant expression, and purification of rGp13 for the first time in Escherichia coli. EMSA data indicated that the purified recombinant Gp13 protein was capable of binding to single-stranded DNA. The protein exhibited maximum binding activity at 32 °C. Furthermore, our bioinformatic analysis has revealed that Gp13 consists of an OB-fold, a characteristic of SSB proteins. However, the arrangement of the OB-fold is unique, being located in the C-terminal domain of Gp13. Despite the importance of SSB proteins in various metabolic processes as well as in various types of PCR, there are no reports on the purification and characterization of SSB proteins from staphylococcal bacteriophages. We expect that the purification and characterization of recombinant Gp13 will help us gain a better insight into its biological activity and make it available in large quantities for molecular biology work.
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Bacteriófagos , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Staphylococcus aureus/genética , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Bacteriófagos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Unión ProteicaRESUMEN
Bacterial biofilm formation by communities of opportunistic bacterial pathogens like Staphylococcus epidermidis is regarded as the primary virulence mechanism facilitating the spread of detrimental nosocomial and implant-associated infections. An 18-kDa small basic protein (Sbp) and its amyloid fibrils account for strengthening the biofilm architecture and scaffolding the S. epidermidis biofilm matrix. Our study reports systematic analysis of the amyloidogenic structural transitions of Sbp and predicts the amyloid core of the protein which may trigger misfolding and aggregation. Herein, we report the novel amyloid inhibitory potential of Camptothecin, a quinoline alkaloid which binds stably to Sbp monomers and redirects the formation of unstructured regions further destabilizing the protein. Molecular dynamics simulations reveal that Camptothecin averts ß-sheet transitions, interrupts with electrostatic interactions and disrupts the intermolecular hydrophobic associations between the exposed hydrophobic amyloidogenic regions of Sbp. Collectively, our study puts forward the first report detailing the heteromolecular associations and amyloid modulatory effects of Camptothecin which may serve as a structural scaffold for the tailored designing of novel drugs targeting the S. epidermidis biofilm matrix.Communicated by Ramaswamy H. Sarma.
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Alcaloides , Quinolinas , Biopelículas , Amiloide/metabolismo , Alcaloides/farmacología , Camptotecina/farmacología , Quinolinas/farmacologíaRESUMEN
Messenger RNA (mRNA) vaccines have been studied for decades, but only recently, during the COVID-19 pandemic, has the technology garnered noteworthy attention. In contrast to traditional vaccines, mRNA vaccines elicit a more balanced immune response, triggering both humoral and cellular components of the adaptive immune system. However, some inherent hurdles associated with stability, immunogenicity, in vivo delivery, along with the novelty of the technology, have generated scepticism in the adoption of mRNA vaccines. Recent developments have pushed to bypass these issues and the approval of mRNA-based vaccines to combat COVID-19 has further highlighted the feasibility, safety, efficacy, and rapid development potential of this platform, thereby pushing it to the forefront of emerging therapeutics. This review aims to demystify mRNA vaccines, delineating the evolution of the technology which has emerged as a timely solution to COVID-19 and exploring the immense potential it offers as a prophylactic option for other cryptic diseases.
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COVID-19 , Pandemias , COVID-19/prevención & control , Humanos , Pandemias/prevención & control , ARN Mensajero/genética , Vacunas Sintéticas/genética , Vacunas de ARNmRESUMEN
In cases involving identification of missing persons, mass disasters and ancient DNA investigations, bone and teeth samples are often the only, and almost always the best, biological material available for DNA profiling. Standard methods for extraction of DNA from such samples involve grinding of the bone and teeth samples. Here, we present an extremely efficient protocol for recovery of DNA from bone samples by a method of scrapping. The study was carried out on 25 samples and it was found that the quantity of DNA isolated by the scrapping method was up to 1.131 ng/µl with a success rate of 93% as compared to a much lower yield of 0.359 ng/µl DNA isolated with a success rate of 28% through the grinding method. The scrapping method of DNA extraction has been proven to be extremely useful in forensic examination of challenging samples that had multiple failures using the traditional grinding method.
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Dermatoglifia del ADN , ADN/análisis , Fémur/química , Repeticiones de Microsatélite , Manejo de Especímenes/métodos , Genética Forense/métodos , Humanos , Reacción en Cadena de la Polimerasa MultiplexRESUMEN
This study evaluated the haplotype diversity of 17 Y chromosomal genetic markers among 202 unrelated males who were randomly selected in the population of Odisha, India. Out of total 196 haplotypes observed in this study, 190 were unique haplotypes. Forensic relevant parameters, viz., gene diversity (GD) and discrimination capacity (DC), were calculated as 0.999999998 and 0.970 respectively, for the studied population. The highest genetic diversity was observed at the locus DYS385a/b (0.9541) and lowest at the locus DYS437 (0.3326) among all the studied Y chromosomal loci. The polymorphic information content (PIC), power of discrimination (PD), and matching probability (PM) was found to be 0.999999965, 0.999999998, and 1.6×10-9 for the tested Y STR loci. The genetic data observed in this study would enrich the existing Y STR data of the Indian population and would also be useful for forensic application.
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Cromosomas Humanos Y , Etnicidad/genética , Variación Genética , Haplotipos , Repeticiones de Microsatélite , Marcadores Genéticos , Genética de Población , Humanos , India/etnología , MasculinoRESUMEN
The bacterial secondary messenger bis-(3',5')-cyclic-dimeric-guanosine monophosphate (c-di-GMP) has been implicated in the pathogenesis of Vibrio cholerae, due to its significant role in regulating the virulence, biofilm formation and motility of the host organism. The VC0395_0300 protein from V. cholerae, possessing a GGEEF sequence has been established as a diguanylate cyclase (DGC) capable of catalyzing the conversion of two GTP molecules to form cyclic-di-GMP. This in turn, plays a crucial role in allowing the organism to adopt a dual lifestyle, thriving both in human and aquatic systems. The difficulty in procuring sufficient amounts of homogenous soluble protein for structural assessment of the GGDEF domain in VC0395_0300 and the lack of soluble protein yield, prompted the truncation into smaller constructs (Sebox31 and Sebox32) carrying the GGDEF domain. The truncates retained their diguanylate cyclase activity comparable to the wild type, and were able to form biofilms as well. Fluorescence and circular dichroism spectroscopy measurements revealed that the basic structural elements do not show significant changes in the truncated proteins as compared to the full-length. This has also been confirmed using homology modeling and molecular docking of the wild type and truncates. This led us to conclude that the truncated constructs retain their activity in spite of the deletions in the N terminal region. This is supportive of the fact that DGC activity in GGDEF proteins is predominantly dependent on the presence of the conserved GGD(/E)EF domain and its interaction with GTP.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Escherichia coli/genética , Liasas de Fósforo-Oxígeno/genética , Vibrio cholerae/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Cólera/microbiología , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Modelos Moleculares , Liasas de Fósforo-Oxígeno/química , Liasas de Fósforo-Oxígeno/metabolismo , Vibrio cholerae/química , Vibrio cholerae/metabolismoRESUMEN
Biofilm formation in Vibrio cholerae empowers the bacteria to lead a dual lifestyle and enhances its infectivity. While the formation and dispersal of the biofilm involves multiple components-both proteinaceous and non-proteinaceous, the key to the regulatory control lies with the ubiquitous secondary signaling molecule, cyclic-di-GMP (c-di-GMP). A number of different cellular components may interact with c-di-GMP, but the onus of synthesis of this molecule lies with a class of enzymes known as diguanylate cyclases (DGCs). DGC activity is generally associated with proteins possessing a GGDEF domain, ubiquitously present across all bacterial systems. V. cholerae is also endowed with multiple DGCs and information about some of them have been pouring in over the past decade. This review summarizes the DGCs confirmed till date in V. cholerae, and emphasizes the importance of DGCs and their product, c-di-GMP in the virulence and lifecycle of the bacteria.
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Proteínas de Escherichia coli , Vibrio cholerae , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biopelículas , GMP Cíclico , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Estilo de Vida , Liasas de Fósforo-Oxígeno/genética , Liasas de Fósforo-Oxígeno/metabolismo , Transducción de Señal , Vibrio cholerae/genética , Vibrio cholerae/metabolismoRESUMEN
In order to find out the genetic diversity in the eastern Indian population of Odisha consisting of various linguistic and ethnic groups, we undertook a study on 508 unrelated healthy individuals belonging to Odisha, India. We assessed genetic variation and compared the data with published literature of Indian population consisting of different ethnic groups from different geographical areas using 21 autosomal STR markers. The most polymorphic and discriminatory STR locus in the studied population was found to be SE33 with the calculated values of 0.94 and 0.991 respectively for both the parameters. The combined power of discrimination (CPD) and combined power of exclusion (CPE) were found to be 1 and 0.999999999704865 respectively. The combined probability of match (CPm) and combined paternity index (CPI) for all 21 autosomal STR loci were found to be 8.01 × 10-26 and 3.45 × 109 respectively. Though, the studied eastern Indian population of Odisha shared its closest genetic affinity with nearest Indian geographical regions, i.e., the population of Jharkhand which is geographically located in eastern India as well as the Central Indian population.
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Etnicidad/genética , Frecuencia de los Genes , Sitios Genéticos , Variación Genética , Repeticiones de Microsatélite , Polimorfismo Genético , Genética de Población , Humanos , India/etnologíaRESUMEN
The mRNA therapeutics is a new class of medicine to treat many various diseases. However, in vitro transcribed (IVT) mRNA triggers immune responses due to recognition by human endosomal and cytoplasmic RNA sensors, but incorporation of modified nucleosides have been shown to reduce such responses. Therefore, an assay signifying important aspects of the human immune system is still required. Here, we present a simple ex vivo method called 'RNA ImmunoGenic Assay' to measure immunogenicity of IVT-mRNAs in human whole blood. Chemically modified and unmodified mRNA are complexed with a transfection reagent (TransIT), and co-incubated in human whole blood. Specific cytokines are measured (TNF-α, INF-α, INF-γ, IL-6 and IL-12p70) using ELISAs. The qPCR analysis is performed to reveal the activation of specific immune pathways. The RNA ImmunoGenic Assay provides a simple and fast method to detect donor specific - immune response against mRNA therapeutics. Graphic abstract: Schematic representation of RNA ImmunoGenic Assay.
RESUMEN
Cyclic-di-GMP (c-di-GMP) synthesized by diguanylate cyclases has been an important and ubiquitous secondary messenger in almost all bacterial systems. In Vibrio cholerae, c-di-GMP plays an intricate role in the production of the exopolysaccharide matrix, and thereby, in biofilm formation. The formation of the surface biofilm enables the bacteria to survive in aquatic bodies, when not infecting a human host. Diguanylate cyclases are the class of enzymes which synthesize c-di-GMP from two molecules of GTP and are endowed with a GGDEF or, a GGEEF signature domain. The VC0395_0300 protein from V. cholerae, has been established as a diguanylate cyclase with a necessary role in biofilm formation. Here we present the structure of an N-terminally truncated form of VC0395_0300, which retains the active GGEEF domain for diguanylate cyclase activity but lacks 160 residues from the poorly organized N-terminal domain. X-ray diffraction data was collected from a crystal of VC0395_0300(161-321) to a resolution of 1.9 Å. The structure displays remarkable topological similarity with diguanylate cyclases from other bacterial systems, but lacks the binding site for c-di-GMP present in its homologues. Finally, we demonstrate the ability of the truncated diguanylate cyclase VC0395_0300(161-321) to produce c-di-GMP, and its role in biofilm formation for the bacteria.
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Proteínas Bacterianas/química , Proteínas de Escherichia coli/química , Liasas de Fósforo-Oxígeno/química , Vibrio cholerae/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Dominio Catalítico , Cristalografía por Rayos X , GMP Cíclico/análogos & derivados , GMP Cíclico/biosíntesis , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Modelos Moleculares , Liasas de Fósforo-Oxígeno/genética , Liasas de Fósforo-Oxígeno/metabolismo , Dominios Proteicos , Sistemas de Mensajero Secundario , Solubilidad , Electricidad Estática , Vibrio cholerae/genética , Vibrio cholerae/fisiologíaRESUMEN
The glycolytic enzyme enolase of Staphylococcus aureus is a highly conserved enzyme which binds to human plasminogen thereby aiding the infection process. The cloning, over expression and purification of S. aureus enolase as well as the effect of various metals upon the catalytic activity and structural stability of the enzyme have been reported. The recombinant enzyme (rSaeno) has been purified to homogeneity in abundant amounts (60 mg/L of culture) and the kinetic parameters (Km = 0.23 +/- 0.013 x 10-3 M; Vmax = 90.98 +/- 0.00052 U/mg) and the optimum pH were calculated. This communication further reports that increasing concentrations of Na+ ions inhibit the enzyme while increasing concentrations of K+ ions were stimulatory. In case of divalent cations, it was found that Mg2+ stimulates the activity of rSaeno while the rest of the divalent cations (Zn2+, Mn2+, Fe2+, Cu2+, Ni2+ and Ca2+) lead to a dose-dependent loss in the activity with a total loss of activity in the presence of Hg2+ and Cr2+. The circular dichroism data indicate that other than Hg2+, Ni2+ and to a certain extent Cu2+, none of the other ions destabilized rSaeno. The inhibitory roles of fluorides, as well as neurotoxic compounds upon the catalytic activity of rSaeno, have also been studied. Conformational changes in rSaeno (induced by ions) were studied using partial trypsin digestion.
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Estabilidad de Enzimas/efectos de los fármacos , Metales/farmacología , Fosfopiruvato Hidratasa/genética , Conformación Proteica/efectos de los fármacos , Catálisis/efectos de los fármacos , Dicroismo Circular , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Iones/química , Iones/farmacología , Metales/química , Fosfopiruvato Hidratasa/química , Fosfopiruvato Hidratasa/aislamiento & purificación , Staphylococcus aureus/enzimología , Staphylococcus aureus/patogenicidadRESUMEN
Since their discovery in the 1990's, the study of a class of non-coding, single-stranded RNAs, christened the microRNAs has opened up new vistas in the field of cancer biology. MicroRNAs bind to their target mRNAs to act as either oncogenes or tumour suppressors. With the near-complete elucidation of the biogenesis pathway, and the advent of rapid sequencing technologies, microRNAs have slowly cemented their place as essential biomarkers for delineating the progression, metastasis, relapse or drug resistance of cancer. Being crucial players in the cancer pathway, there has been considerable urgency in designing molecules - both at the nucleotide and non-nucleotide level to counter the effects of their binding. A number of different approaches have yielded quite a body of compounds which have been found to be effective in the treatment of various tumours across many different organs. In this study, the focus is on the review of the timeline of discovery and characterization of microRNAs, underlining their importance in different cancers, shedding light on the discovery of anti-microRNA compounds and illustrating their uses in deriving new strategies to combat cancer.
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Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , MicroARNs/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Animales , Proliferación Celular/efectos de los fármacos , Humanos , MicroARNs/metabolismo , Neoplasias/patologíaRESUMEN
We report the biological activity of three Cu(II) complexes [Cu(pabt)Cl] (1), [Cu(pma)Cl] (2), and [Cu(pdta)Cl]Cl (3) (pabt = N-(2-mercaptophenyl)-2'-pyridylmethylenimine, pma = N-(2-pyridylmethyl)-2-mercaptoaniline, pdta = 2,2'-di(pyridyl-2-methyleneimine)diphenyl disulfide). 1-3 display four-line EPR multiplet in solution at RT suggesting that these are mononuclear. DNA-binding studies using spectrophotometric titration of these complexes with calf thymus DNA showed binding through intercalation mode which was found to be consistent with the observation of increased viscosity of DNA and quenching of fluorescence of ethidium bromide bound DNA in the presence of these complexes. All three complexes were found to be efficient in bringing about oxidative and hydrolytic cleavage of DNA. The proposed mechanism of hydrolytic DNA cleavage has been discussed. MTT assay showed remarkable cytotoxicity on cervical cancer HeLa cell line and the IC50 values were 1.27, 4.13, and 3.92 µM for 1, 2 and 3, respectively, as compared to the IC50 value (13 µM) reported for cisplatin in HeLa cells. AO/PI and Annexin-V/PI assay suggest the induction of cell death primarily via apoptotic pathway. Nuclear staining using DAPI was used to assess changes in nuclear morphology during apoptotic cell death. The role of reactive oxygen species (ROS) for induction of apoptotic cell death was studied using H2DCF-DA assay and the result suggests that the generation of ROS by the complexes may be a possible cause for their antiproliferative activity. TUNEL assay showed DNA fragmentation in apoptotic cells. Cell cycle analysis using flow cytometry showed significant increase in the G2/M phase in HeLa cells by the compounds 1-3. Mononuclear Cu(II) complexes display remarkable cytotoxicity against cervical cancer HeLa cell line. The generation of ROS by the complexes may be a cause of their antiproliferative activity. Fluorescent images from DAPI staining assay revealed that the cells undergoing apoptosis displayed typical features like cell shrinkage, membrane blebbing, chromatin condensation and nuclear fragmentation. TUNEL assay showed DNA fragmentation in apoptotic cells.
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Antineoplásicos/farmacología , Complejos de Coordinación/farmacología , Cobre/química , ADN/química , Bases de Schiff/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/toxicidad , Bovinos , Proliferación Celular/efectos de los fármacos , Complejos de Coordinación/síntesis química , Complejos de Coordinación/química , Complejos de Coordinación/toxicidad , División del ADN/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Células HEK293 , Células HeLa , Humanos , Hidrólisis , Sustancias Intercalantes/síntesis química , Sustancias Intercalantes/química , Sustancias Intercalantes/farmacología , Sustancias Intercalantes/toxicidad , Ligandos , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismo , Bases de Schiff/síntesis química , Bases de Schiff/química , Bases de Schiff/toxicidad , ViscosidadRESUMEN
BACKGROUND: Cyclic-di-GMP (c-di-GMP) is a ubiquitous secondary messenger molecule in bacteria synthesized by diguanylate cyclases. This universal messenger regulates diverse cellular functions in bacteria at the transcriptional, translational and posttranslational levels. The cellular functions regulated by c-di-GMP include cell motility, cell cycle progression, virulence, biofilm formation, antibiotic production and other unknown functions. The VC0395_0300 protein from the chromosome I of the Vibrio cholerae classical strain O395, serotype O1 has been established to be a diguanylate cyclase with a necessary role in biofilm formation. OBJECTIVE: Mutations in the central position of the GGEEF active site of VC0395_0300 protein have been created by site-directed mutagenesis. The conditions for maximum production of mutated protein have been optimized. While there is a significant loss-of-biofilm-forming activity in the mutants, the basis for the same needed an investigation at the structural level. METHODS: Subsequently, the mutant proteins have been characterized using spectrofluorimetry and circular dichroism spectroscopy. RESULTS: While the unfolding pattern of the mutant proteins shows some changes with respect to the wild type, the overall structure of the protein does not show significant changes due to the mutagenesis, despite the absence of biofilm formation in the mutants. CONCLUSION: This led us to conclude that whatever changes that occur in the mutated proteins, do not disturb the GGEEF domain architecture, but are restricted to the local architecture, and are hence, subtle in nature.
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Proteínas Bacterianas/genética , Biopelículas , Proteínas de Escherichia coli/genética , Liasas de Fósforo-Oxígeno/genética , Vibrio cholerae/genética , Vibrio cholerae/fisiología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Mutagénesis Sitio-Dirigida , Liasas de Fósforo-Oxígeno/química , Liasas de Fósforo-Oxígeno/metabolismo , Conformación ProteicaRESUMEN
BACKGROUND: The Gp07 protein of aureophage Phi11 exhibits growth inhibitory effects when overexpressed in E. coli .The protein harbors two domains- an amino terminal Bro-like domain and a carboxy terminal Ant superfamily like KilA domain, of which the KilA domain retains the growth inhibitory effect of Gp07. METHODS: We studied the effects exerted by the overexpression of Gp07 and its separate domains upon the growth rate as well as the morphology of the E. coli cells. Additionally, we generated a mutant of Gp07 (designated as ΔGp07) by deleting the first eleven amino acid residues from the amino-terminal region of Gp07, and studied its growth inhibitory effects upon E. coli. RESULTS: Our results indicate that Gp07, ΔGp07 as well as the Carboxy-terminal region of Gp07 upon overexpression, retards the growth rate of the E. coli cells and also induces filamentation in the cells. Surprisingly, our data clearly suggests that the growth inhibition and filamentation induced by the the amino-terminal domain of Gp07 is temporal in nature. CONCLUSION: The carboxy-terminal of domain of gp07 is essential for its activity.